In this large, prospective pregnancy cohort, we analyzed the association between mid-pregnancy vaginal microbiome, race, and sPTB. We found that vaginal microbiomes were significantly associated with sPTB, race, douching and other maternal factors. Many of these maternal factors, like poverty, education, marital status, age, douching and race, have stronger associations with the vaginal microbiome than the vaginal microbiome has with sPTB (Fig. 1c). Consistent with previous studies 10,27, we found that the vaginal microbiomes of Black and White women were significantly different, with higher alpha diversity, higher abundance of L. iners and lower abundance of L. crispatus for Black women. The microbial difference between sPTB and term controls is mainly driven by a higher L. crispatus abundance in term controls, similar to previous reports 13,14. Because of the strong intercorrelations between maternal factors such as race, poverty, education, marital status and douching (Fig. S2), we stratified the dataset by race and douching with the aim of uncovering potentially stronger sPTB microbial signatures that are independent of race and douching.
With the community state types assigned based on the most abundant taxon, the sPTB risk associated with L. crispatus dominated community state is about 60% of that for L. iners dominated microbiome (Fig. 2d, 13% and 22% in all participants, and 3.5% and 5.9% when oversampling of cases in this nested case-control design is accounted for). The alpha diversity of L. crispatus dominated microbiome is significantly lower than that of L. iners dominated microbiome, indicating that L. crispatus may suppress the colonization and development of BV-like microbiome while L. iners does not. Compared to L. crispatus, vaginal microbiome dominated by L. iners also more often shift towards a diverse community 28. For example, L. iners enhance the adhesion of Gardnerella spp. to cervical epithelial cells, and Gardnerella spp. displaced adherent L. crispatus but not L. iners from epithelial cells 29. Previous research also suggests that L. crispatus and Gardnerella were exclusive while L. iners and Gardnerella often co-exist 14. This may explain the higher sPTB risk associated with L. iners dominated microbiome compared to L. crispatus in our population. The sPTB risk associated with community state “Lacto_other” dominated by other Lactobacillus species (mostly L. gasseri and L. jensenii/fornicalis/psittaci) is also significantly higher than L. crispatus dominated community state, indicating that these species were also not as protective as L. crispatus.
With a similar number of Black and White participants, we analyzed the associations between microbiome and the sPTB risk in each race separately and found that risk of sPTB associated with L. crispatus and L. iners are similar for Black and White women (Fig. 2g). Although at the US population level, black women have substantially higher risk of PTB 17, in the PIN study specifically, black race is only marginally associated with PTB (OR 1.3, 95% CI 1.0, 1.6) 18. Our findings, that race does not modify the association between L. crispatus and L. iners and sPTB, may suggest that the disparity in PTB rates at the population level may in part be due to the lower prevalence of L. crispatus dominated microbiome among Black women. While the associations between L. crispatus, L. iners, and sPTB are independent of race, we could not verify whether risk associated with other community patterns are also consistent between races, because of their relatively low prevalence among participants. Future studies with a larger number of participants are needed to further investigate other taxa.
Douching is often associated with BV 30–32, although it is difficult to determine whether douching increases the risk of BV or BV leads to douching. In this study, we found that douching played an important role in the structure of the vaginal microbiome. Among women who did not douche, Black and White women have different microbiome. Specifically, White women had a notably higher abundance of L. crispatus, lower abundance of L. iners and higher abundance of other Lactobacillus species. However, among women who did douche, the microbiomes of Black and White women were similar, and featured lower abundance of L. crispatus and higher abundance of L. iners. It was reported that the genome of L. iners AB-1 contains genes that could contribute to its survival in an environment of fluctuating conditions, including Fe-S cluster protein for oxidative stress, alkaline shock and universal stress proteins 28. L. iners also has a stronger ability to adhere to human fibronection than other vaginal bacteria strains such as L. crispatus ATCC 3800 33. Thus, it is possible that douching habits influence L. iners dominated microbiome less than L. crispatus dominated microbiome. At the same time, the percentage of sPTB cases was higher in douching groups in both Black and White women although causality cannot be directly inferred. It is possible that a pre-existing dysbiotic state caused both douching behavior and sPTB, or alternatively, that douching disrupted the healthier L. crispatus dominated microbiome that then shifted to a higher risk microbiome, ultimately leading to sPTB. Future studies with longitudinal vaginal track sampling, and longitudinal information on douching behavior, would be required to disentangle these interconnected features. Regardless, our results suggest that douching behavior is significantly associated with the vaginal microbiome, and that race-related differences in vaginal microbiome are erased in the population of women that report douching.
In summary, in this prospective study of mid-pregnancy microbiome and sPTB in a well characterized cohort of Black and White women, we found that the vaginal microbiome of Black women was characterized by higher diversity, lower abundance of L. crispatus, and higher abundance of L. iners. These differences were obscured once maternal douching behavior was considered—specifically, among women who douche, there were no material differences in microbiome by race. Additionally, we found that women with microbiome dominated by L. crispatus had lower risk of sPTB, and women with microbiome dominated by L. iners had higher risk of sPTB, and these associations were the same for Black and White women. To our knowledge, this is the first study of the vaginal microbiome and sPTB to consider the influence of douching, and we found that douching has a significant influence on the vaginal microbiome that should be considered in future studies.
Finally, it is important to note that while we present differences in microbial community patterns by race to be consistent with the prior literature 10,11, we observed strong inter-correlations across a number of maternal factors whose effects cannot easily be separated. These intercorrelated factors include race, poverty level, psychosocial stress, education, marital status, and maternal age. In this as with many other medical research studies, maternally self-classified race only crudely captures complex social determinants of health 34, and thus disparities in microbial community patterns that we observe in relation to race may actually result from factors such as but not limited to her diet, her access to high quality medical care, her social support and life experiences, her psychosocial stress, and her experiences of discrimination. These pathways, that may explain differences in vaginal microbial community patterns by race, need further investigation and elucidation. Although this is one of the largest studies of the associations between vaginal microbiome and sPTB, it still lacks power for analyzing less abundant microbes and whether the combination of race or other social factors and douching influence the consistency of microbial signatures. Pooled studies across cohorts with similar metagenomics data may enable a more precise investigation of rare species as well as the influence of maternal factors that may explain or modify effects of vaginal microbiome on sPTB.